3-Chloro-1,3-oxazines or oxazolidines as antibacterial agents

ABSTRACT

There is provided, novel 3-chloro-tetrahydro-1,3-oxazine or oxazolidine compounds, exhibiting antibacterial activity and being of low chlorine potential of the formula: ##STR1## wherein each of R 1  and R 2 , which may be the same or different, represent an alkyl group of from 1 to 20 carbon atoms (C 1  -C 5  being preferred); wherein each of R 5  and R 6 , which may be the same or different, represent a hydrogen atom or an alkyl group of from 1 to 20 carbon atoms (C 1  -C 5  being preferred); wherein l represents an integer of 1 or 2 and wherein each of R 3  and R 4 , which may be the same or different, represent an alkyl group of from 1 to 20 carbon atoms (C 1  -C 5  being preferred) a --(CH 2 ) n  X group, wherein n represents an integer of from 1 to 20 and wherein X represents a dimethylamino group, a diethylamino group, a trimethylammonium group, a triethylammonium group, a dimethylammonium group, a diethylammonium group, a --COOR 7  group, a --OOCR 8  group, and a --OR 9  group, wherein each of R 7  through R 9 , respectively, represent an alkyl group of from 1 to 5 carbon atoms or a benzyl group. In addition, R 3  and R 4  can also represent a ##STR2## group, wherein Y represents a --(CH 2 ) n  --W--(CH 2 ) m  -- group or a &gt;CH--Z group, wherein W represents a --O-- atom, a --CH 2  -- group, a &gt;NCH 3  -- group, a &gt;NHCH 3   +   group, a &gt;NC 2  H 5  group, a &gt;NHC 2  H 5   +   group, a &gt;N(CH 3 ) 2   +   group, or a &gt;N(C 2  H 5 ) 2   +  group, wherein n is the same or different from m and wherein each of n and m represent an integer of from 0 to 2; and wherein Z is defined in accordance with X above.

CROSS-REFERENCE TO RELATED APPLICATONS

This application is a divisional application of our earlier co-pendingapplication, Ser. No. 566,747, filed Apr. 10, 1975 now U.S. Pat. No.3,954,985, which in turn was a divisional application of still earlierco-pending application, Ser. No. 456,744, filed Apr. 1, 1974, now U.S.Pat. No. 3,897,425.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to novel antibacterial low chlorinepotential compounds and more specifically, the present invention isdirected to a totally new class of such compounds, termed3-chloro-tetrahydro-1,3-oxazines or oxazolidines as describedhereinafter.

2. Description of the Prior Art

It is known in the art that certain N-chloro-2-oxazolidinones possessantibacterial activity. However, a review of the literature concerningsuch compounds will readily reveal that these compounds are higherchlorine potential compounds and as a result thereof, while suchcompounds can exhibit a sufficient antibacterial activity, their higherchlorine potential imparts to these compounds, a "bleach" capability.That is, due to their high chlorine potential, theN-chloro-2-oxazolidinones, while capable of controlling bacterialgrowth, will also tend to be more corrosive. Consequently, for the mostpart, these compounds have been employed as bleaching agents. See, U.S.Pat. No. 3,591,601.

SUMMARY OF THE INVENTION

In view of the foregoing, it is readily apparent that there is a greatneed to develop suitable antibacterial agents of low chlorine potential,which exhibit sufficient antibacterial activity and yet, are lesscorrosive.

Therefore, it is one object of the present invention to developantibacterial agents of low chlorine potential, which exhibit sufficientantibacterial activity.

It is a second object of the present invention to develop antibacterialagents which in addition to exhibiting sufficient antibacterial activityand of being of low chlorine potential, also fail to be relativelycorrosive.

Finally, it is a third object of the present invention to developantibacterial agents which meet the above criteria and yet, arenon-persistant. That is, compounds which, in aqueous media, exhibitsufficient antibacterial activity over a short time span, afterwhich,decomposition of the compound occurs.

Accordingly, with the foregoing in mind, the present invention isdirected to a novel class of low chlorine potential compounds, whichexhibit antibacterial activity and are deemed non-persistant, whichcompounds have the following formula: ##STR3## wherein each of R₁ andR₂, which may be the same or different, represent an alkyl group of from1 to 20 carbon atoms (C₁ -C₅ being preferred); wherein each of R₅ andR₆, which may be the same or different, represent a hydrogen atom or analkyl group of from 1 to 20 carbon atoms (C₁ -C₅ being preferred);wherein l represents integer of 1 or 2 and wherein each of R₃ and R₄,which may be the same or different, represent an alkyl group of from 1to 20 carbon atoms (C₁ -C₅ being preferred), a --(CH₂)_(n) X group,wherein n represents an integer of from 1 to 20 and wherein X representsa dimethylamino group, a diethylamino group, a trimethylammonium group,a triethylammonium group, a dimethylammonium group, a diethylammoniumgroup, a --COOR₇ group, a --OOCR₈ group, a --OR.sub. 9 group, whereineach of R₇ through R₉, respectively, represent an alkyl group of from 1to 5 carbon atoms or a benzyl group. In addition, R₃ and R₄ can alsorepresent a ##STR4## group, wherein Y represents a --(CH₂)_(n)--W--(CH₂)_(m) -- group or a>CH-Z group, wherein W represents an--O--atom, a --CH₂ -- group, a>NCH₃ -- group, a>NHCH₃ ⁺ group, a>NC₂ H₅group, a>NHC₂ H₅ ⁺ group, a>N(CH₃)₂ ⁺ group, or a>N(C₂ H₅)₂ ⁺ group,wherein n is the same or different from m and wherein each of n and mrepresent an integer of from 0 to 2; and wherein Z is defined inaccordance with X above.

In the case where R₅ and R₆ may represent an alkyl group of from 1 to 20carbon atoms or a hydrogen atom, the hydrogen atom form is preferred.This is true even when R₅ and R₆ represent a carbon atom range of from 1to 5.

Where feasible, applicants prefer to use the proton salt (HX salt),wherein X represents a pharmaceutically acceptable anion derived from apharmaceutically acceptable acid addition salt of the compound, thoughuse of the free base is quite acceptable. The proton salts can easily beprepared by simply reacting the free base compound with apharmaceutically acceptable acid, such as hydrochloric acid, hydrobromicacid, methanesulfonic acid, and the like.

At this point, it should be emphasized that the term "antibacterial" asemployed in this application also includes "antifungal" activity aswell.

The compounds of the present inventon are readily prepared by thesynthesis procedure outlined below, wherein R₁, R₂, R₃, R₄, R₅, R₆ andl, are defined as above. ##STR5## The above reaction scheme is carriedout under standard temperature and pressure. With respect to step (1),the solvent employed is one which is capable of removing water from thefirst reaction step as it is formed. Without limitation, illustrativesolvents capable of achieving this function are benzene, toluene, orxylene. As an alternative embodiment, a desiccant, such as magnesiumsulfate or a molecular sieve can be employed to remove water formedduring the reaction.

As for step (2), any conventional chlorinating agent can be employed andillustrative of such agents, suitable for applicants' purpose is NaOCl,Ca(OCl)₂, t-BuOCl, N-chlorosuccinimide and chlorine. Naturally, theaforementioned chlorinating agents are only illustrative of the widevariety of conventional chlorinating agents suitable for applicants'purpose and it is believed that this is understood by the skilledartisan concerned with the subject matter of this invention.

A better understanding of the present invention will be gained from areview of the following examples, which are simply illustrative andnon-limitative thereof.

EXAMPLE I Preparation of3-chloro-2,2,4,4-tetramethyl-tetrahydro-1,3-oxazolidine

Firstly, the precursor compound (2,2,4,4-tetramethyl-1,3-oxazolidine wasprepared. To 237 g (3.0 mole) of 2-amino-2-methyl-1 -propanol, there wasadded approximately 750 ml of dry benzene containing 174 g (3.0 mole) ofacetone.

A few crystals of para-toluenesulfonic acid were added to the reactionmixture and the solution was stirred under a Dean-Stark water separatorat relux temperature. When the theoretical amount of water wascollected, the reaction mixture was distilled at atmospheric pressure.The 2,2,4,4-tetramethyl-tetrahydro-1,3-oxazolidine was collected as aclear, colorless distillate, bp 128°-130° C. 220 g (1.7 mole), 57%yield.

Analysis Calculated for: C₇ H₁₅ NO: C, 65.07; H, 11.70; and N, 10.84.Found: C, 65.34; H, 11.80; and N, 11.00.

Next, the final compound(3-chloro-2,2,4,4-tetramethyl-tetrahydro-1,3-oxazolidine) was preparedfrom the precursor material. To 175 ml of 0.65 M sodium hypochlorite(0.11 mole) at 0° C., there was added dropwise with stirring, theprecursor compound obtained earlier, while the reaction mixture wasmaintained between a pH of from 4 to 6 through the use of 1M HCl.

After 30 minutes at 0° C., the reaction mixture was extracted withdichloromethane and the extracts were combined and dried over anhydroussodium sulfate. Following filtration, the dichloromethane was removedunder reduced pressure and the3-chloro-2,2,4,4-tetramethyl-tetrahydro-1,3-oxazolidine was isolated asa pale yellow liquid, bp 65°-67° C. (12 mm), 10.1 g (0.062 mole), a 56%yield.

Analysis Calculated for: C₇ H₁₄ ClNO: C, 51.37; H, 8.62; N, 8.56 and Cl,21.7. Found: C, 51.36; H, 8.77; and Cl, 19.2.

Once the final compound was prepared, it was subjected to antibacterialand stability studies as described in Tables 1 and 2, set forth on thefollowing pages.

                                      TABLE 1                                     __________________________________________________________________________                               CONCENTRATION DATA                                                                          GERMICIDAL ACTIVITY TIME (MIN)                    CONDITIONS            POSI- BACTERIA                             COMPOUND     pH     :                                                                              DILUENT                                                                             COMPOUND                                                                             ;                                                                              TIVE Cl                                                                             12228                                                                             10536                                                                             10031                                                                             9027                                                                              6538                                                                             4617              __________________________________________________________________________     ##STR6##    0.1M NaOAc pH 4.6 0.1M NaOAc  pH 4.6                                                 : :                                                                            H.sub.2 O Serum                                                                     8.26 ×  10.sup.-.sup.3 M 1355 ppm 8.26                                  × 10.sup.-.sup.3 M 1355                                                        ; ;                                                                            289 ppm 289 ppm                                                                     0.5 >10                                                                           0.5 3                                                                             0.5 >10                                                                           0.5 4                                                                             0.5 >10                                                                          0.5 6              ##STR7##    0.1M NaH.sub.2 PO.sub.4 pH 7.00 0.1M NaH.sub.2 PO.sub.4 pH                    7.00   : :                                                                            H.sub.2 O Serum                                                                     8.92 × 10.sup.-.sup.3 M 1463 ppm 8.92                                   × 10.sup.-.sup.3 M  1463                                                       ; ;                                                                            312 ppm 312 ppm                                                                     1 6 0.5 3                                                                             0.5 5                                                                             1 7 3 >10                                                                            1 >10              ##STR8##    0.1M Na.sub.2 B.sub.4 O.sub.7 pH 8.8 0.1M Na.sub.2 B.sub.4                    O.sub.7 pH 8.8                                                                       : :                                                                            H.sub.2 O Serum                                                                     8.13 × 10.sup.-.sup.3 M 1333 ppm 8.13                                   × 10.sup..sup.-3 M 1333                                                        ; ;                                                                            285 ppm  285 ppm                                                                    >5 >10                                                                            2 4 4 9 2 4 >5 >10                                                                           3 8               __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________    STABILITY ANALYSIS OF 3-CHLORO-2,2,4,4-TETRAMETHYL-1 TETRAHYDRO-1,3-OXAZOL    IDINE                                                                          ##STR9##                                                                     CONDITIONS                                                                    Buffer    pH     T (° C)                                                                       Initial concentration of 3                                                                    Half-life (hr.)                       __________________________________________________________________________    0.1 M NaOAc                                                                             pH 4.6 40     1.88 × 10.sup.-.sup.3 M                                                                 2.2                                                           2.59 × 10.sup.-.sup.3 M                                                                 2.2                                                           5.47 × 10.sup.-.sup.3 M                                                                 2.6                                   0.1 M NaH.sub.2 PO.sub.4                                                                pH 7.0 40     1.68 × 10.sup.-.sup.3 M                                                                 ˜70                                                     3.05 × 10.sup.-.sup.3 M                                                                 ˜54                                                     6.70 × 10.sup.-.sup.3 M                                                                 ˜54                             0.1 M Na.sub.2 B.sub.4 O.sub.7                                                          pH 9.3 40     1.52 × 10.sup.-.sup.3 M                                                                 ˜5.0                                                    2.49 × 10.sup.-.sup.3 M                                                                 ˜4.6                                                    5.98 × 10.sup.-.sup.3 M                                                                 ˜3.3                            H.sub.2 O               6.23 × 10.sup.-.sup.3 M                                                                 ˜58                             __________________________________________________________________________

example ii preparation of3-chloro-2,2-[spiro-1'-methyl-4'-piperidinyl]-4,4-dimethyl-1,3-oxazolidine and its HX salts

Firstly, the precursor compound,2,2-[spiro-1'-methyl-4'-piperidinyl]-4,4-dimethyl-1,3-oxazolidine wasprepared. To 44.5 g (0.5 mole) of 2-amino-2-methyl-1-propanol, there wasadded approximately 400 ml of dry benzene containing 56.5 g (0.5 mole)of 1-methyl-4-piperidone. The solution was stirred under a Dean-Starkwater separator at reflux temperature.

When the theoretical amount of water was collected, the benzene wasremoved under reduced pressure to yield a brown oil. The precursorcompound was collected as a clear, colorless distillate, bp 63°-65° C.(1.2 mm), 58.9 g (0.32 mole), 64% yield.

Next, the final compound 3-chloro-2,2-[spiro-1'-methyl-4'-piperidinyl]-4,4-dimethyl-1,3-oxazolidine was prepared from theprecursor material in the manner described. To 50 ml of 0.65 M sodiumhypochlorite (0.03 mole) at 0° C., there was added with stirring 5.52 g(0.03 mole) of the precursor compound obtained earlier.

After 30 minutes at 0° C., the pale yellow solid was isolated byfiltration, washed thoroughly with cold water and then dried in vacuoover calcium sulfate to give3-chloro-2,2-[spiro-1'-methyl-4'-piperidinyl]-4,4-dimethyl-1,3-oxazolidine,mp 46°-48° C., sublimation at 35° C. (0.5 mm); UV (H₂ O) λ max 265 nm,ε=270 M.sup.⁻¹ cm.sup.⁻¹.

Analysis Calculated for: C₁₀ H₁₉ ClN₂ O: C, 54.91; H, 8.76; N, 12.81;and Cl, 16.2. Found: C, 54.77; H, 8.90; N, 12.85; and Cl, 15.1.

PREPARATION OF THE HX SALTS

The methanesulfonate salt of the above-isolated compound was prepared bysimply reacting the free base with an ethereal solution ofmethanesulfonic acid, mp 124°-125° C. (dec).

Analysis Calculated for: C₁₁ H₂₃ ClN₂ O₄ S: C, 41.96; H, 7.36; N, 8.90;and Cl, 11.3. Found: C, 41.27; H, 7.41; N, 8.49; and Cl, 10.6

In similar fashion, the hydrochloride salt of the above-isolatedcompound was prepared as follows. To an ethereal solution containing 1.1g (0.005 mole), at 0° C., there was added dropwise with stirring, 2 mlof 2.26 M HCl/Et₂ O, diluted to approximately 25 ml using anhydrousether, (0.0045 mole). The suspension was maintained at 0° C. for 30minutes and the white solid which formed was isolated by filtration andthoroughly washed with anhydrous ether under an atmosphere of nitrogen.The solid isolated was dried in vacuo over calcium sulfate to give thecorresponding hydrochloride salt, mp 120 °-121° C. (dec.), 1.18 g(0.0046 mole), 93% yield.

Analysis Calculated for: C₁₀ H₂₀ Cl₂ N₂ O: C, 47.06; H, 7.90; N, 10.98;Cl, 13.9. Found: C, 44.39; H, 7.56; N, 9.91; and Cl, 13.1.

Tables 3 and 4, set forth on the following pages, illustrate theantibacterial activity and stability values obtained from the free-baseand the methanesulfonate salt prepared by the procedure described inExample II. Tables 5 and 6, set forth on the following pages, illustratethe stability values determined for the hydrochloride andmethanesulfonate salts of the free-base compound prepared by theprocedure of Example II.

                                      TABLE 3                                     __________________________________________________________________________    ANTIBACTERIAL ACTIVITY OF 3-CHLORO-2,2-[-METHYL-4'-PIPERIDINYL]-4,4-DIMETH    YL-1,3-OXAZOLIDINE                                                                             Conditions                                                                    Buffer/ Concentration Data                                                                          Antibacterial Activity, Time                                                  (min.)                                 Compound         pH  Diluent                                                                           Compound                                                                              Positive Cl                                                                         12228                                                                             10536                                                                             10031                                                                             9025                                                                             6538                                                                             4617                                                                             6501              __________________________________________________________________________     ##STR10##       0.1 M NaOc pH 4.6 0.1 M NaOAc pH 4.6                                              H.sub.2 O Serum                                                                   6.52 × 10.sup..sup.-3 M 1428 ppm 5.69                                   × 10.sup..sup.-3 M 1246 ppm                                                     231 ppm 202 ppm                                                                     1 >10                                                                             0.5 2                                                                             0.5 0.5 > 10                                                                         5 >10                                                                            -- --                                                                            0.5 3              ##STR11##       0.5 M NaOAc pH 4.6 0.5 M NaOAc pH 4.6                                             H.sub.2 O Serum                                                                   21.00 × 10.sup.-.sup.3 M 4578 ppm 20.50                                 × 10.sup.-.sup.3 M 4469 ppm                                                     745 ppm 727 ppm                                                                     0.5 2                                                                             0.5 0.5                                                                           0.5 0.5                                                                           0.5 1                                                                            0.5 3                                                                            0.5 1                                                                            0.5 0.5            ##STR12##       0.5 M NaOAc pH 4.6 0.5 M NaOAc pH 4.6                                             H.sub.2 O Serum                                                                   17.0 × 10.sup..sup.-3 5355 ppm 17.0                                     × 10.sup..sup.-3 5355 ppm                                                       603 ppm 603 ppm                                                                     0.5 2                                                                             0.5 0.5                                                                           0.5 1                                                                             0.5 1                                                                            1 6                                                                              0.5 1                                                                            0.5 1             .sup.-OSO.sub.2 CH.sub.3                                                      __________________________________________________________________________

                                      table 4                                     __________________________________________________________________________    antibacterial activity of 3-chloro-2,2-[spiro-1'-methyl-4'-piperidinyl]-4,    4-dimethyl-1,3-oxazolidine                                                                     conditions                                                                             Concentration Data                                                                         Antibacterial Activity, Time                                                  (min.)                                                  Buffer/          Posi-                                       Compound         pH   Diluent                                                                           Compound                                                                              tive Cl                                                                            12228                                                                             10536                                                                             10031                                                                             9027                                                                             6538                                                                             461                                                                              6501              __________________________________________________________________________     ##STR13##       0.5 M NaH.sub.2 PO.sub.4 pH 7.0 0.5 M NaH.sub.2 PO.sub.4                      H 7.0                                                                              H.sub.2 O Serum                                                                   20.75 × 10.sup.-.sup.3 M 4525 ppm 19.88                                 × 10.sup..sup.-3 M 4334                                                         736 ppm 705 ppm                                                                    1 5 0.5 1 8 1 10                                                                             1 >10                                                                            0.5 5                                                                            1 7                ##STR14##       0.5 M NaH.sub.2 PO.sub.4 pH 7.0 0.5 M NaH.sub.2 PO.sub.4                      H 7.0                                                                              H.sub.2 O                                                                         19.75 × 10.sup.-.sup.3 M 6221 ppm 19.13                                 × 10.sup.-.sup.3 M 6026                                                         700 ppm 678 ppm                                                                    4 >10                                                                             1 4 1 >10                                                                             1 >10                                                                            4 >10                                                                            1 >10                                                                            1 >10             .sup.-OSO.sub.2 CH.sub.3                                                      __________________________________________________________________________

                  table 5                                                         ______________________________________                                        stability of 3-chloro-2,2-[spiro-1'-methyl-4'-                                piperidinal 4,4-dimenthyl-1,3-oxazolidine                                     hydrochloride in the neat state at 40° c                               ______________________________________                                        time(days)                                                                              Wt. (mg).sup.a                                                                             V.sub.T (ml).sup.b                                                                        %Cl.sup.C                                  ______________________________________                                        0         29.11        22.60       13.7                                                 48.50        35.95       13.1                                       1         35.15        24.50       12.3                                                 81.29        57.20       12.5                                       2         39.95        27.75       12.3                                                 40.30        27.05       11.9                                       3         39.28        27.70       12.5                                                 80.50        55.60       12.2                                       4         39.25        26.75       12.1                                                 94.08        65.30       12.3                                       5         77.55        53.90       12.3                                                 61.72        43.20       12.4                                       7         45.32        31.25       12.2                                                 49.26        33.75       12.1                                       10        50.71        34.20       11.9                                                 46.87        31.15       11.8                                       14        40.44        27.55       12.1                                                 38.32        25.54       11.8                                       21        66.61        45.00       12.0                                                 68.78        47.00       12.1                                       28        40.16        26.10       11.5                                                 46.41        30.40       11.6                                       35        57.21        38.05       11.8                                                 51.65        33.95       11.6                                       51        25.50        13.70        9.5                                       ______________________________________                                         .sup.a Weight of sample analyzed.                                             .sup.b Volume of 10.sup.-.sup.2 N sodium thiosulfate required to titrate      the sample at time (T).                                                       .sup.c Percentage of positive chlorine in the sample analyzed.           

                  TABLE 6                                                         ______________________________________                                        STABILITY OF 3-CHLORO-2,2-[SPIRO-1'-METHYL-4'                                 PIPERIDINIUM 1,3-OXAZOLIDINE                                                  METHANESULFONATE IN THE NEAT STATE AT 40° C                            ______________________________________                                        Time (days)                                                                             Wt. (mg).sup.a                                                                             V.sub.T (ml).sup.b                                                                        %Cl.sup.c                                  ______________________________________                                        0         74.29        44.35       10.6                                                 58.10        34.70       10.6                                       1         36.27        21.00       10.2                                                 60.50        35.10       10.3                                       2         34.77        20.15       10.3                                                 74.49        41.45       9.8                                        3         155.01       85.95       9.8                                                  21.46        12.10       10.0                                       4         119.17       63.35       9.4                                        5         117.89       64.35       9.7                                                  72.92        39.90       9.7                                        7         37.76        19.85       9.3                                                  159.15       86.80       9.7                                        10        146.99       81.70       9.8                                        14        109.44       54.65       8.8                                                  43.93        22.40       9.0                                        21        138.91       73.90       9.4                                        28        42.54        22.65       9.4                                                  69.21        35.60       9.1                                        35        114.65       57.70       8.9                                                  47.36        24.05       9.0                                        51        46.23        20.80       8.0                                        ______________________________________                                         .sup.a Weight of sample analyzed.                                             .sup.b Volume of 10.sup.-.sup.2 N sodium thiosulfate required to titrate      the sample at time (T).                                                       .sup.c Percentage of positive chlorine in the sample analyzed.           

ANTIBACTERIAL ACTIVITY STUDIES

The procedure employed to determine the antibacterial activity of3-chloro-2,2,4,4-tetramethyl-tetrahydro-1,3-oxazolidine and3-chloro-2,2-[spiro-1'-methyl-4'-piperidinyl]-4,4-dimethyl-1,3-oxazolidinewas based primarily on a modification of the serial dilution method ofanalysis. However, instead of determining the minimum inhibitoryconcentration parameters for the compound investigated, applicantsalternatively chose to determine the bactericidal end-point for a givenconcentration of the compound investigated. Consequently, applicants'studies were established to determine the time required for completesterilization of the micro-organism tested, when exposed to a givenconcentration of the compound investigated.

The method and reagents employed in applicants' antibacterial studiesare described below:

    ______________________________________                                                                Overnight Broth                                       Organism     ATCC Code  Culture(Organisms/ml                                  ______________________________________                                        Staph. epidermidis                                                                         12228      5 × 10.sup.6                                    E. coli      10536      10 × 10.sup.6                                   Kleb. pneumoniae                                                                           10031      12 × 10.sup.6 - 13 × 10.sup.6             Pseud. aeruginosa                                                                           9027      12 × 10.sup.6 - 13 × 10.sup.6             Staph. aureus                                                                               6538      6 × 10.sup.6 - 8 × 10.sup.6               Bord. bronchiseptica                                                                        4617      3 × 10.sup.6                                    ______________________________________                                    

Nutrient Broth B.B.L. -- 8 g/1000 ml distilled water. The broth contains5 g gelysate peptone and 3 g beef extract. The solution has a pH of 6.9.Nutrient Agar -- 23 g/1000 ml. distilled water. The nutrient contains 5g gelysate peptone, 3 g beef extract and 15 g agar.

Horse Serum T.C. -- 10% horse serum solution in distilled water. Theserum solution was freshly prepared and adjusted to a pH of 7 usingcarbon dioxide prior to its use.

METHOD

A stock solution of each compound identified above was prepared using anappropriate buffered solution.

For screening in the absence of a denaturing agent (e.g., horse serum)an equal volume of distilled water and the resulting solution wassubjected to the screen.

For screening in the presence of a denaturing agent, a volume of thestock solution was diluted using an equal volume of 10% horse serum.When necessary, the final solution was adjusted to the desired pH using1N HCl and the solution was permitted to stand at room temperature forthirty minutes prior to the screening procedure.

To 5 ml of the stock solution being evaluated, there was added 0.2 ml ofan overnight broth culture containing the particular micro-organismbeing investigated (see above). At time intervals of 0.5, 1, 2, 3, 4, 5,. . . minutes, a loop of this suspension was subcultured into 5 ml of asterile nutrient broth. All the samples were then incubated at 37° C.for seven days with daily observation for evidence of bacterial growth.The time interval reported is for that sample in which no bacterialgrowth was observed after the incubation period.

Aside from the foregoing, several controls were also employed asdescribed below.

CONTROL 1

This control was designed basically to insure viability of the overnightbroth culture.

To 5 ml of a sterile 0.9% sodium chloride solution, there was added 0.2ml of an overnight broth culture containing the particularmicro-organism being investigated. A loop of this suspension wassubcultured into 5 ml of a sterile nutrient broth and incubated at 37°C. for seven days.

CONTROL 2

This control was designed to insure that the diution factor of thenutrient broth was beyond any bacteriostatic activity of each compound(as identified above) tested.

To 5 ml of a sterile nutrient broth there was added a loop of a solutionof each compound as described above and the solution was mixedimmediately. To this soluton, there was then added a loop of anovernight broth culture which was diluted 25× with a 0.9% sodiumchloride solution. Incubation was carried out for 7 days at atemperature of 37° C.

CONTROL 3

This control was employed to insure the bacterial growth observed wasthat due to the organism being tested, rather than contanimation from aforeign organism.

At the same time intervals used for subculturing the test solution intonutrient broth during the screening procedure, a loop of the testsolution was also subcultured onto sterile nutrient agar plates. Thistechnique was useful for observing the characteristic colonialmorphology of each organism.

CONTROL 4

This control was used initially to insure that the pH of the solutionand the concentration of the buffer species did not inhibit thebacterial growth during the time intervals used in the screeningprocedure.

The entire screening procedure was conducted for each buffered solutionusing the buffered solution rather than the solution of each testedcompound in the procedure.

By following the reaction scheme as illustrated in Examples I and II,all the compounds of the present invention can be prepared.

While all compounds encompassed within applicants' generic formula domeet applicants' criteria, i.e., exhibit sufficient antibacterial andantifungal activity with low chlorine potential and remainnon-persistant, still, certain compounds are preferred. These compoundsare:

1. 3-chloro-2,2,4,4-tetramethyl-tetrahydro-1,3-oxazolidine,

2. 3-chloro-2,2,4,4-tetramethyl-tetrahydro-1,3-oxazine,

3.3-chloro-2-methyl-2-(1-diethylamino-3-propyl)-4,4-dimethyl-1,3-oxazolidineor its HX salt,

4.3-chloro-2-methyl-2-(1-diethylamino-3-propyl)-4,4-dimethyl-tetrahydro-1,3-oxazineor its HX salt,

5.3-chloro-2,2[spiro-1'-methyl-4'-piperidinyl]-4,4-dimethyl-1,3-oxazolidineorits HX salt,

6. 3-chloro-2-methyl-2-(diethylaminoethyl)-4,4-dimethyl-1,3-oxazolidineor its HX salts,

7. 3-chloro-2-methyl-2-(diethylaminomethyl)-4,4-dimethyl-1,3-oxazolidineor its HX salts

These compounds are conveniently used in aqueous solution. They may beapplied by any conventional means, e.g., spray, wipe, etc.

Although the present invention has been adequately described in theforegoing specification and examples included therein, it is obviouslyapparent that various changes and/or modifications can be made theretowithout departing from the spirit and scope thereof.

What we claim is:
 1. A method for inhibiting bacterial growth which comprises treating said bacterial growth with an antibacterially effective amount of a compound of the formula: ##STR15## wherein each of R₁ and R₂ which may be the same or different represents an alkyl group of from 1 to 20 carbon atoms; wherein each of R₅ and R₆ which may be the same or different, represents a hydrogen atom or alkyl of from 1 to 20 carbon atoms; and wherein each of R.sub. 3 and R₄ which may be the same or different represent ##STR16## wherein Y represents --(CH₂)_(n) --W--(CH₂)_(m) -- or >CH-Z, wherein W represents a member selected from the group consisting of -- O-- , --CH₂ --, >NCH₃, >NHCH₃ ⁺, >NC₂ H₅, >NHC₂ H₅ ⁺, >N(CH₃)₂ ⁺, and >N(C₂ H₅)₂ ⁺, wherein n is the same or different from m and wherein each of n and m represent an integer of from 0 to 2 and wherein Z represents a member selected from the group consisting of dimethylamino, diethylamino, trimethylammonium, triethylammonium, dimethylammonium, diethylammonium, --COOR₇, --OOCR₈ and --OR₉, wherein each of R₇ through R₉ respectively, represent a member selected from the group consisting of alkyl of from 1 to 5 carbon atoms and benzyl; and wherein l represents an integer of 1 or
 2. 2. The method of claim 1, wherein R₁ and R₂ represent an alkyl group of from 1 to 5 carbon atoms.
 3. The method of claim 1, wherein R₅ and R₆ represent an alkyl group of from 1 to 5 carbon atoms.
 4. The method of claim 1, wherein R₅ and R₆ represent a hydrogen atom.
 5. The method of claim 1, wherein said compound is:3-Chloro-2,2-[spiro-1'-methyl-4'-piperidinyl]-4,4-dimethyl-1,3-oxazolidine and its HX salt, wheren X represents a pharmaceutically acceptable anion. 